A sensitive fluorescence anisotropy method for detection of lead (II) ion by a G-quadruplex-inducible DNA aptamer

Abstract

h i g h l i g h t s• A fluorescence anisotropy approach for detection of Pb 2+ was developed.• The strategy was based on bindinginduced allosteric conformational change of aptamer probe.• The sensing mechanism was established by testing the photoinduced electron transfer interaction. in homogeneous solution by a G-rich thrombin binding aptamer (TBA). The TBA labeled with 6-carboxytetramethylrhodamine (TMR) at the seventh thymine nucleotide was used as a fluorescent probe for signaling Pb 2+ . It was found that the aptamer probe had a high FA in the absence of Pb 2+ . This is because the rotation of TMR is restricted by intramolecular interaction with the adjacent guanine bases, which results in photoinduced electron transfer (PET). When the aptamer probe binds to Pb 2+ to form G-quadruplex, the intramolecular interaction should be eliminated, resulting in faster rotation of the fluorophore TMR in solution. Therefore, FA of aptamer probe is expected to decrease significantly upon binding to Pb 2+ . Indeed, we observed a decrease in FA of aptamer probe upon Pb 2+ binding. Circular dichroism, fluorescence spectra, and fluorescence lifetime measurement were used to verify the reliability and reasonability of the sensing mechanism. By monitoring the FA change of the aptamer probe, we were able to real-time detect binding between the TBA probe and Pb 2+ . Moreover, the aptamer probe was exploited as a recognition element for quantification of Pb 2+ in homogeneous solution. The change in FA showed a linear response to Pb 2+ from 10 nM to 2.0 M, with 1.0 nM limit of detection. In addition, this sensing system exhibited good selectivity for Pb 2+ over other metal ions. The method is simple, quick and inherits the advantages of aptamer and FA.

Metal Cations in G-Quadruplex Folding and Stability

“…Another divalent ion dependent folding GQ was discovered by Shafer and co-workers in thrombin binding aptamer which folds into an intramolecular GQ in the presence of Pb 2+ (Smirnov and Shafer, 2000b ). The precision and high affinity binding of thrombin aptamer with Pb 2+ ions was later utilized to develop biosensor for the Pb 2+ ion detection (Li et al, 2011 ; Jacobi et al, 2012 ; Brenneman et al, 2013 ; Zhang D. et al, 2014 ). Several attempts have been made to understand the structural details of divalent cation binding to GQs over the monovalent ion binding.…”

Phosphate-perylene modified G-quadruplex probes for the detection of Pb2+ using fluorescence anisotropy

“…In addition, a thrombin binding aptamer (TBA) sequence with a fluorophore-quencher pair (FAM and DABCYL at 5 0 and 3 0 termini) could form a G-quadruplex with Pb 2+ , which induced the increase of FRET response. 34 However, the current covalent attachment of a State Key Laboratory of Natural and Biomimetic Drugs, fluorophores to G-quadruplex oligonucleotides caused the decrease in the binding affinity and/or selectivity of the modified G-quadruplexes for the target analytes. 29,30 Many small molecules and ions, such as ATP, 31,32 L-histidine 33 and Pb 2+ 34,35 were detected when forming complexes with fluorophore-labeled oligonucleotide probes to induce the change of fluorescence anisotropy signals.…”

Spinach RNA aptamer detects lead(ii) with high selectivity

“…Our Spinach sensor offers the following distinct advantages: it does not need fluorophore-quencher pairs that require covalent attachment to the nucleic acid, does not involve expensive nanomaterials or complicated detection systems that have been used for some previous DNA-based Pb 2+ sensors, 16 and has a very simple design and operation that is cost effective and easy to use for ‘on the spot’ detection of Pb 2+ .…”